The present invention relates to novel cross-linking agents, more particularly to novel biodegradable cross-linking agents. Earlier use of cross-linking agents in a variety of fields involving proteins, carbohydrates or polymers is well established. Even biodegradable cross-links have previously been prepared and utilized. However, none before have utilized particular and advantageous cross-linker designs of the present invention.
Within the pharmaceutical, agricultural, veterinary, and environmental industries, much attention has been directed to the applications of biodegradable polymers. The Oxford English dictionary defines biodegradable as: xe2x80x9csusceptible to the decomposing action of living organisms especially bacteria or broken down by biochemical processes in the body.xe2x80x9d However, due to the advent of the widespread use of polyhydroxyacids as degradable polymers, this definition should be extended to include non-enzymatic chemical degradation which can progress at an appreciable rate under biologically relevant conditions (the most relevant condition being water at pH 7; 100 mM salt and 37xc2x0 C.). Thus, the meaning of the term biodegradation can be broadened to include the breakdown of high molecular weight structures into less complicated, smaller, and soluble molecules by hydrolysis or other biologically derived processes.
In the biomaterials/pharmaceutical area, there is great interest in the use of biodegradable materials in vivo, due to performance and regulatory requirements. However, most of the reports on biodegradable materials have focused on linear water-insoluble hydroxyacid polyesters. Much less work has been done on biodegradable network polymers which are cross-linked. Therefore, due to the unique properties of network polymers, it is to be expected that biodegradable networks will find many new and important applications
Much work has been accomplished in the last 20 years in the area of hydrophobic biodegradable polymers, wherein the biodegradable moieties comprise esters, lactones, orthoesters, carbonates, phosphazines, and anhydrides. Generally the polymers made of these biodegradable linkages are not water soluble and therefore in themselves are not amenable for use in systems where water is required, such as in hydrogels.
Since the mechanism of biodegradation in these polymers is generally through the hydrolytically-active components of water (hydronium and hydroxide ions), the rate of hydrolytic scission of the bonds holding a polymer network together is generally pH sensitive, with these moieties being susceptible to both specific-acid catalyzed hydrolysis and base hydrolysis. Other factors affecting the degradation of materials made of these polymers are the degree of polymer crystallinity, the polymer volume fraction, the polymer molecular weight, the cross-link density, and the steric and electronic effects at the site of degradation.
Biodegradable network structures are prepared by placing covalent or non-covalent bonds within the network structure that are broken under biologically relevant conditions. This involves the use of two separate structural motifs. The degradable structure is either placed into (i) the polymer backbone or (ii) into the cross-linker structure. The method described herein creates a degradable structure through placing degradable regions in the cross-linking domain of the network. One of the first occurrences of degradable hydrogels was published in 1983 by Heller. This system contains a water soluble linear copolymer containing PEG, glycolylglycolic acid and fumaric acid linkages. The fumaric acid allowed the linear polymer to be cross-linked through free radical polymerization in a second network forming polymerization step, thus creating a polymer network which could degrade through hydrolysis of the glycolic ester linkages. This is an example of creating degradable linkages in the polymer backbone.
The first truly degradable cross-linking agents were made from aryl diazo compounds for delivery of drugs in the digestive tract. The diazo moiety is cleaved by a bacterial azoreductase which is present in the colon. This has been used to create colon specific delivery systems (Brondsted et al. and Saffan et al.). Another biodegradable cross-linking agent appears in the work of Ulbrich and Duncan where a bis-vinylic compound based on hydroxyl amine was synthesized. Hydrogels made from this degradable cross-linker were shown to undergo hydroxide induced hydrolysis of the nitrogen-oxygen bond.
Hubbell et al. have made hydrogels composed of macromonomers composed of a central PEG diol which was used as a bifunctional alcohol in the tin octanoate catalyzed transesterifying ring opening polymerization of lactide to give a bis-oligolactate PEG. This compound was then reacted with acryloyl chloride to give a macromolecular cross-linker which could be formed into a homo-polymer interpenetrating network of PEG and oligolactylacrylate through free radical polymerization (Pathak et al.). Hubbell mostly intended these compounds for use as photopolymerizable homo-polymers useful to prevent surgical adhesions.
A second solution to this problem has been recently reported in the work of Van Dijk et al. which is the first report of a biodegradable cross-linking macromonomer composed of alpha-hydroxy esters (Van Dijk-Wolthius et al.). This work combines natural polymers with synthetic polymers in an interpenetrating network. This group functionalized dextran with oligo-alpha hydroxy acid domains which were end capped with vinyl regions that were polymerized into biodegradable networks via free radical polymerization.
The most recent report of a biodegradable cross-linking agent was one designed to undergo enzymatic degradation. This cross-linker is composed of a centro-symmetric peptide terminated by acrylamide moieties with a central diamine linking the two ends (Kurisawa et al.). This report is related to the invention described herein in that the property of biodegradability is built into the polymer network by first synthesizing a small symmetrical cross-linker which can undergo cleavage, then incorporating this in a polymer network.
Since degradability is a kinetic effect, the properties of degradable gel networks are the similar to those standard gel networks, except they change with time. The two main properties that are exhibited by degradable hydrogel networks are swelling and network porosity that increase with time as the network degrades.
The main feature observed with degradable cross-linked polymer networks in solvents which cause them to swell is that the polymer network swells as it degrades. This is because network degradation results in a decrease in cross-link density. As the cross-link density decreases there is more available volume for solvent within the network. The solvent increasingly permeates the network structure, driven by a favorable thermodynamic mixing of solvent with the polymer network.
Important uses envisioned for degradable gels are as controlled drug delivery devices and as degradable polymers for other in vivo uses. These devices are able to change from a high viscosity material (gel) to a lower viscosity soluble material (sol). The resulting water soluble linear polymer can then be readily transported and excreted or degraded further.
Degradable hydrogel networks offer the opportunities to effect the diffusitivity of materials bound in the hydrogel network, because as the network degrades the diffusion coefficient of molecules in the network increases with time thus facilitating the release of materials locked within the polymer network (Park).
Moreover, because the hydrogel network structure itself is of such a high molecular weight, transport of the hydrogel network out of the body or environment is slow. This is especially true in vivo where non-degradable implanted hydrogel networks can remain in the body for many years (Torchilin et al.). Therefore, such devices would be more useful if they could be made of a high molecular weight polymer that would degrade into smaller molecular weight components after the device has performed its task and then could be excreted through normal routes of clearance.
Since excretion of polymers is molecular weight-dependent (Drobnik et al.), with the preferred route being through the renal endothelia (Taylor et al. and Tomlinson), the chains making up the polymer backbone should be between 10 and 100 kDa. Because the material is engineered to degrade into excretable parts, biodegradable hydrogel networks offer increased biocompatibility.
Biodegradable network polymers can be used as carriers for biologically active substances. These include proteins, peptides, hormones, anti-cancer agents antibiotics, herbicides, insecticides and cell suspensions. The hydrophilic or hydrophobic polymer network can act as a stabilizing agent for the encapsulated species and as a means to effect a controlled release of the agent in to the surrounding tissue or systemic circulation. By changing the size of the depot, the degree of porosity, and the rate of degradation (through modification of the degradable regions in the polymer network) controlled release depots with a variety of release characteristics can be fashioned for application in the medical and diagnostic areas.
Owing to the ability of hydrophilic network polymers to adsorb water, biodegradable versions of these networks may prove to have many uses in items for example, sanitary napkins, wound dressings, and diapers. When these materials are used in consort with other degradable materials a completely biodegradable and disposable product could be produced. Although a literature search in the Chemical Abstracts database for biodegradable adsorbents produced no citations, the use of degradable adsorbents in the above mentioned products would be very desirable.
There is a great need for biodegradable adhesives and sealers in surgery and elsewhere. Synthetic polymers have been used as adhesives in surgery with the cyano acrylate esters being the most commonly cited. Recent reports using biodegradable networks as sealants in dentistry and orthopedics have displayed the utility of biodegradable polymers (Burkoth). Here the use of a biodegradable cross-linking monomer (bis-methacrylated diacid anhydride) which has been photopolymerized is envisioned for use in dentistry. Here a hydrophobic network-forming monomer is photopolymerized in situ to form a mechanically stable and non-swellable bonding material. Degradability would be a desirable property for any short term application and of course would be undesirable for long term applications.
Since most biodegradable polymers are not soluble in water, a hydrophilic drug is formulated in these polymers by a dispersion method using a two phase system of water (containing drug) and organic solvent (containing the polymer). The solvent is removed by evaporation resulting in a solid polymer containing aqueous droplets. This type of system suffers from the need to use organic solvents which would be undesirable for protein delivery since the solvent may denature the protein. Therefore it is envisioned that hydrophilic biodegradable network polymers will improve the range of drugs delivered from this general glass of polymers.
The use of nanoparticles for colloidal drug delivery has been a goal of formulation scientists for the last 20 years. Nanoparticles are defined as any solid particle between 10 and 1000 nm and are composed of natural or more commonly synthetic polymers. The most useful method of production for the lower end of this size range is emulsion polymerization, where micelles act as a reaction template for the formation of a growing polymer particle. For passive delivery of anticancer agents to tumors, nanometer size particles (50-200 nm) are required. The small size is required for extravasation of the nanoparticles through the permeable tumor vasculature in a process termed the EPR effect (enhanced permeability and retention) (Duncan).
Another important feature of any nanocarrier is the biocompatibility of the particle. This requires that the polymer particle degrades after some period so that it may be excreted. These criteria require polymer compositions that are well tolerated. To date there are no reports in the literature of degradable nanogels composed of well-tolerated parenternal polymers.
Hydrogel particles can be made in several sizes according to the performance requirements of the drug delivery system being engineered. Gel particles in the nanometer size range that are capable of being retained in tumor tissue are preferred for delivery of anticancer agents. Methods for the creation of approximately 100 nm in diameter hydrogel particles involve the use of surfactant-based emulsion polymerizations in water. To make ionomeric nanogels by this method it is necessary to include a hydrophobic component in the monomer mixture, thus allowing partitioning of the monomers into the micellar phase followed by particle nucleation and further monomer adsorption (normally emulsion polymerizations are used to make hydrophobic latexes).
Another important consideration is the means by which the carrier will load the drug substance to be delivered. The loading capacity of non-ionic hydrogels is generally limited by the aqueous solubility of the drug. However if the drug is charged, groups of opposite charge to the drug can be incorporated into the polymer to allow high drug loading through ion exchange. An interesting and perhaps useful property resulting from inclusion of charged monomers in the polymer network is a pH induced volume response of the polymer.
To date most biodegradable polymers have been synthesized using stepwise condensation of monomer resulting in a polydispersed molecular architecture. Since the rate of degradation is in part directly related to this architecture, this method results in the undesirable property that the material will contain cross-links with a variety of degradation rates. Secondly, since synthetic biodegradable polymers are generally water insoluble, there is a need for degradable moieties that are readily incorporated into water soluble monomers or polymers. Biodegradable moieties based on the non-soluble degradable units can be combined with water soluble oligomeric regions or polymers, resulting in a biodegradable structure.
Therefore as an object of the present invention the new material would have the preferred characteristics that it was easily synthesized, composed of biocompatible components, and have a well defined molecular structure leading to defined biodegradation rates.
It is a further object of the present invention that it be easily incorporated in many different polymer processing options such as polymer microparticles, nanoparticles and slab gels.
Therefore, the use of organic synthesis methodology to incorporate monodispersed degradable sequences into the monomer structure before polymer formation permits control of overall degradation as well as the release rate of entrapped substances.
Previous work in the area of creating biodegradable cross-linkers by Hubbel teaches a method to create degradable sequences using ring opening polymerization of lactide or glycolide. This method creates a mixture of degradable units with varying molecular weights or chain lengths in the end product. The present invention described herein teaches a method of stepwise synthesis of the degradable region which creates a pure compound at the end of the synthesis. Therefore, since the length of the degradable region will be the major structural determinant of the degradation rate, the present invention provides for a more controlled degradation rate than the Hubbel invention. Our invention also provides compounds which will be easier to purify than the Hubbel invention owing to stepwise syntheses of the degradable region and the resulting purity of the reaction product. Other advantages of our invention over Hubbel""s invention are that the invention described herein is applicable to hydrophobic networks as well as hydrophilic networks whereas Hubbel is restricted to hydrophilic networks, and the invention herein can generate all useful properties such as rapid degradation rate and water solubility through the syntheses of oligomeric cross-linking compounds without resorting to polymeric cross-linking compounds.
The present preferred embodiment of this invention is as cross-linkers which are composed of a symmetrical diacid attached to at least one biodegradable region. These regions may consist of alpha hydroxy acids such as glycolic or lactic acid. These degradable portions are then terminated directly or indirectly by a functional group which may be polymerized. Moreover component pieces of the degradable gel such as lactic, glycolic and succinic acids are members of the Krebs cycle and therefore readily metabolized in vivo, while the end groups become incorporated into water-soluble polymer, which is eliminated by renal excretion.
In one important aspect the present invention concerns a monomeric or oligomeric cross-linker comprising a polyacid core with at least two acidic groups directly or indirectly connected to a reactive group usuable to cross-link polymer filaments, with at least one acidic group being connected to a region degradable under aqueous conditions and the degradable regions or (in the case of a single degradable region), the degradable region at at least one other acidic group directly or indirectly having a covalently attached reactive group usable to cross-link polymer filaments interceding between the acidic group and a reactive group. Thus the at least two reactive groups are always interspaced by at least one degradable region. In many preferred applications, the cross-linker is utilized to cross-link water soluble polymeric filaments. The polyacid core may be attached to a water soluble region that is in turn attached to a degradable region (or vice versa) having an attached reactive group. A polycarboxylic acid is the preferred polyacid. The polyacid core is preferably a diacid, triacid, tetraacid or pentaacid. The most preferred polyacid core is a diacid. Preferred polyacids or polycarboxylic acids. Alkyl-based diacids such as malonic, succinic, adipic, fumaric, maleic, sebacic and tartaric are preferred. Diacids such as succinic, adipic or malonic acid are particularly preferred. A triacid such as citric acid, for example, is usable. Tetra-and penta-acids such as ethylenediamine tetraacidic acid (EDTA) or diethylenetramine pentaacetic acid (DTPA) are usable, for example. When cross-linked polymer filaments are formed according to the present invention, they are cross-linked by a component having at least one degradable region. Preferred degradable regions include poly(alpha-hydroxy acids), although other hydroxy alkyl acids that may form polyesters can be used to form biodegradable regions. Preferred polyesters include those of glycolic acid, DL lactic acid, L lactic acid, oligomers, monomers or combinations thereof. Cross-linkers of the present invention may also include a degradable region containing one or more groups such as anhydride, a orthoester and/or a phosphoester. In certain cases the biodegradable region may contain at least one amide functionality. The cross-linker of the present cross-linker may also include an ethylene glycol oligomer, oligo(ethylene glycol), poly(ethylene oxide), poly(vinyl pyrolidone), poly(propylene oxide), poly(ethyloxazoline), or combinations of these substances.
Preferred reactive groups are those that contain a carbon-carbon double bond, a carbonate, a carbamate, a hydrazone, a hydrazino, a cyclic ether, acid halide, a acylazide, succinimidyl ester, imidazolide or amino functionality. Other reactive groups may be used that are known to those skilled in the art to be precursors to polymers.
Utilizing the cross-linkers of the present invention, networks of polymer filaments may be formed by thermal, catalytic or photochemical initiation. Networks of polymer filaments may likewise be formed by pH changes. Networks of polymer filaments may also be formed for example by free radical addition or Michael addition.
The present invention comprises a network of polymer filaments formed by precipitation or emulsion polymerization and cross-linked by a monomeric or oligomeric cross-linker comprising a poly acid core with at least one acidic group connected to a region degradable under in vivo conditions and having at least two covalently attached reactive groups usable to cross-link polymer filaments. Polymeric filaments to be cross-linked include preformed polymer filaments such as polynucleic acids, polypeptides, proteins or carbohydrates. Such cross-linked polymeric filaments may be utilized to contain biologically active molecules. The biologically active molecules may be organic molecules, proteins, carbohydrates, polynucleic acids, whole cells, tissues or tissue aggregates.
The preferred monomeric or oligomeric cross-linker of the present invention has a polyacid core with a molecular weight between about 60 and about 400 Daltons. The degradable region(s) has a preferable molecular weight range of about 70 to about 500 Daltons. The reactive groups of the cross-linker of the present invention may be end groups and have preferred molecular weights between about 10 and 300 Daltons.
An important aspect of the present invention is a monomeric or oligomeric cross-linker comprising a polyacid core with at least two esterified groups being connected (directly or indirectly) to reactive groups usable to cross-link polymer filaments. Between at least one reactive group and polyacid core is a region degradable under aqueous conditions. Thus the cross-linker is usable to form cross-linked polymer filaments. In a preferred embodiment, the polyacid core has two acidic groups connected to a region degradable under aqueous conditions, each having a covalently attached reactive group usable to form cross-linked polymer filaments. In certain cases the cross-linkers of the present invention may contain a water soluble region located between at least one carboxyl group and its associated reactive group. A preferred polymer filament for cross-linking is a hydrogel. In certain cases the polymer filament being cross-linked may be hydrophobic.
In many cases the polyacid core of the present inventive cross-linker is a diacid, such as for example succinic acid, adipic acid, fumaric acid, maleic acid, sebacic acid or malonic acid. Triacids such as citric acid are also usable. Other triacids will be apparent to those of skill in the art. Tetraacids and pentaacids may also be used. A preferred tetraacid is ethylene diamine tetraacetic acid (EDTA) and a preferred pentaacid is diethylenetriamine pentaaceticic acid (DTPA).
Acids that may be used as a polyacid core include citric acid, tartaric acid and the like. A preferred biodegradable region for use in the cross-linkers of the present invention is one that comprises a hydroxy alkyl acid ester. A preferred hydroxy acid ester is an alpha hydroxy acid ester. Under some circumstances the degradable region may be a peptide. Preferred degradable polyesters include glycolic polyester, DL lactic acid polyester and L lactic acid ester or combinations thereof. In certain cases the degradable region of the cross-linker of the present invention may comprise an anhydride, orthoester or phosphoester linkages. In certain cases the reactive group of the present inventive cross-linker contains a carbon-carbon double bond. In some cases the reactive group is an end group, e.g. at the end of a degradable region. The reactive group may also contain a carbonate, carbamate hydrazone, hydrazino, cyclic ether, acid halide, acyl azide, succinimidyl ester, imidazolide or amino functionality.
The cross-linker of the present invention may be utilized to form networks of polymer films formed by thermal catalytic or photochemical initiation. In certain cases networks of polymer films may be formed as induced by a pH change and then cross-linked. In other cases, networks of polymer films may be formed through reactions involving free radical addition or Michael addition. The aqueous conditions under which the cross-linkers of the present invention are degradable are most frequently physiological conditions.
In an important aspect, the present invention comprises a network of polymer filaments formed by precipitation, dispersion or emulsion polymerization and cross-linked by a monomeric or oligomeric cross-linker having a polyacid core with at least two esterified groups connected to a covalently attached reactive group used to cross-link polymer filaments and at least one acidic group having a region degradable under aqueous conditions between the acidic group and the reactive group.
Also included in the present invention are networks of polymer filaments of polynucleic acids, polypeptides, proteins or carbohydrates and cross-linked by a monomeric or oligomeric cross-linker comprising a polyacid core with at least two esterified groups connected to at least one region degradable under in vivo conditions, and having a covalently attached reactive group cross-linking the polymer filaments.
In both cases of networked polymer filaments, these networks may contain biologically active molecules. Because the cross-links are degradable, these biological molecules will be expected to be released.
In one important aspect, the present invention comprises a network of polymer filaments cross-linked by a monomeric or oligomeric cross-linker comprising a polyacid core with at least two acidic groups connected to at least one region degradable under in vivo conditions, and both acidic groups connected to a covalently attached reactive group and defined further as comprising an organic molecule, inorganic molecule, protein, carbohydrate, poly(nucleic acid), cell, tissue or tissue aggregate.
Additionally, the invention includes a network of polymer filaments cross-linked by monomeric or oligomeric cross-linker comprising a central polyacid core with at least two acidic groups connected to at least one region degradable under in vivo conditions, and terminated by a covalently attached reactive end group usable to cross-link polymer filaments, the network comprising an organic radioisotope, inorganic radioisotope or nuclear magnetic resonance relaxation reagent.
According to the present invention the polyacid core has a preferred molecular weight between about 60 and about 400 daltons. The degradable region of the cross-linker has a preferred molecular weight between about 70 and about 500 daltons. The reactive groups of the present invention generally have molecular weights between about 10 and about 300 daltons.